Substituted tetracycline compounds as antifungal agents

ABSTRACT

Methods and compositions for treating fungal associated disorders in subjects are discussed.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/097,457, filed Mar.14, 2002, entitled “Substituted Tetracycline Compounds as AntifungalAgents,” Issuing; which claims priority to U.S. Provisional ApplicationSer. No. 60/275,948, filed Mar. 14, 2001, entitled “SubstitutedTetracycline Compounds as Antifungal Agents”. The entire contents ofeach of these applications is hereby incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

For many years, the development of effective therapeutic agents forfungal diseases (mycoses) has lacked the attention devoted to drugseffective against other infective organisms. The most common mycoticinfections are superficial in nature, are not life threatening, andprovide little medical impetus to pharmaceutical companies to developnovel treatments. This scenario is changing, however, and while deathfrom fungal disease is not new, the incidence of systemic fungalinfections that cause these fatalities is increasing. Ironically,advances in modern medical techniques in other fields (immunosuppressiveand/or cytotoxic therapy) and the advent of disease such as AcquiredImmuno Deficiency Syndrome (AIDS) are major contributing causes to theincreased number of serious fungal infections.

Fungal associated disorders can, thus, be divided into thelife-threatening systemic infections, such as histoplasmosis, systemiccandidiasis, aspergillosis, blastomycosis, coccidioidomycosis,paracoccidioidomycosis, and cryptococcosis, and the more commonsuperficial ones, such as dermatophyte (ringworm) infections, forexample, tinea pedis (athlete's foot) and tinea cruris (jock itch),candidiasis, and actinomycosis. The life-threatening fungal infectionsare a growing problem not only for immunosuppressed or immunocompromisedindividuals as noted above but individuals with other viral infections,such as cytomegalovirus (CMV), and influenza, for cancer patientsreceiving chemotherapy or radiotherapy, for transplant patientsreceiving antirejection agents, and for patients that have receivedtoxic chemicals, metals and radiation exposure.

Mycoses are often caused by fungi which are opportunists, rather thanpathogens. Candidiasis, aspergillosis, phycomycosis, nocardiosis, andcryptococcosis are typically opportunistic fungal infections. Forexample, Candida albicans, is normally found in the alimentary tract asa commensal, yet it is a major cause of systemic fungal infections inimmunocomprised patients and topical infections in healthy individuals.

Most drugs currently available for the treatment of mycoses have limitedefficacy or are poorly tolerated. A persistent and vexatious problemwith antifungal agents, largely unattended by the prior art, is the lackof an agent that is easy and economical to synthesize, and possesseshigh activity and broad spectrum activity against organisms, lowtoxicity and limited adverse effects.

Moreover, many known agents merely have fungistatic properties, ratherthan fungicidal properties. Fungistatic activity is the ability toprevent growth of fungi, while fungicidal (fungitoxic) activity is theability to kill the fungi. Many agents used in the treatment ofsuperficial mycoses are virtually devoid of either fungistatic orfungicidal actions in the concentrations used, and their beneficialeffects probably depend upon factors not related to any direct effect onfungi.

Despite a plethora of agents which have or are alleged to haveantifungal properties, most are simply fungistatic and not fungitoxic.For those that are fungicidal, for example, amphotericin B, there aresevere adverse side effects which limit their use and their chemicalproperties, e.g., solubility, limit drug delivery method.

SUMMARY OF THE INVENTION

Although opportunistic systemic fungal infections have a high morbidityand mortality and their incidence is increasing, the art has yet toprovide a safe, effective water soluble, simple-to-synthesize,fungitoxic agent with a broad antifungal spectrum of activity coupledwith limited adverse effects and low toxicity.

In one embodiment, the invention pertains, at least in part to a methodfor inhibiting the growth of a fungus. The method includes contactingthe fungus with an effective amount of a substituted tetracyclinecompound, such that the growth of said fungus is inhibited. In a furtherembodiment, the substituted tetracycline compound is of formula I:

-   -   X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;    -   R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen,        alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen,        or hydrogen;    -   R³, R¹¹ and R¹² are each hydrogen, or a pro-drug moiety;    -   R¹⁰ is hydrogen, a prodrug moiety, or linked to R⁹ to form a        ring;    -   R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl,        aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,        alkyl carbonyloxy, or aryl carbonyloxy;    -   R⁶ and R^(6′) are each independently hydrogen, methylene,        absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or        an arylalkyl;    -   R⁷ is hydrogen, halogen, nitro, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,        amino, arylalkenyl, arylalkynyl, or        —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a);    -   R⁹ is hydrogen, halogen, nitro, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,        amino, arylalkenyl, arylalkynyl, thionitroso(e.g., —N═S), or        —CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);    -   Z is CR^(9d)R^(9e), S, NR^(9b) or O;    -   Z′ is O, S, or NR^(9f);    -   W is CR^(7d)R^(7e), S, NR^(7b) or O;    -   W′ is O, NR^(7f) S;    -   R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(9a), R^(9b), R^(9c),        R^(9d), and R^(9e) are each independently hydrogen, acyl, alkyl,        alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl,        alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,        alkylamino, or an arylalkyl;    -   R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl; and    -   Y′ and Y are each independently hydrogen, halogen, hydroxyl,        cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl, and pharmaceutically acceptable salts thereof.

The invention also pertains to a method for treating a fungal associateddisorder in a subject. The method includes administering to the subjectan effective amount of a substituted tetracycline compound such that thesubject is treated for the fungal associated disorder.

The invention also pertains to pharmaceutical compositions, whichcontain an effective amount of a substituted tetracycline compound totreat a fungal associated disorder in a subject and a pharmaceuticallyacceptable carrier.

The invention also pertains to a method of killing fungus, by contactingthe fungus with a substituted tetracycline compound of the invention,such that the fungus is killed.

DETAILED DESCRIPTION OF THE INVENTION

In addition to their well known antibacterial properties, minocyclineand doxycycline have been shown to possess limited antifungal activityboth alone and in synergy with amphotericin B (Antimicrob. AgentsChemother. (1984), 26(6)837-40; Pathol Biol. (1975) 23(9):725-8). Theinvention pertains, at least in part, to methods and pharmaceuticalcompositions comprising substituted tetracycline compounds with enhancedantifungal activity.

In an embodiment, the invention pertains to methods for inhibiting thegrowth of a fungus. The method includes contacting the fungus with aneffective amount of a substituted tetracycline compound, such that thegrowth of the fungus is inhibited.

The terms “fungus” or “fungi” include a variety of nucleated,sporebearing organisms which are devoid of chlorophyll. The termincludes all fungi whose growth can be inhibited by the compounds of theinvention. Examples include, but are not limited to, yeasts, mildews,molds, rusts, and mushrooms. Examples of fungi also include, but are notlimited to Aspergillus fumigatus, Aspergillus flavus, Aspergillusnidulans, Candida albicans, Candida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis, Cryptococcus neoformans, Issatchenkia orientalis,Coccidioides, Paracoccidioides, Histoplasma, Blastomyces, and Neurosporacrassa. In one embodiment, the fungi of the invention includes fungi ofthe genus Candida (e.g., C. tropicalis, C. parapsilosis, C. lusitaniae,C. krusei, C. guilliermondii, C. glabrala, C. dubliniensis, and C.albicans).

The term “inhibiting the growth of a fungus” includes both fungistaticand fungicidal activity. Fungistatic activity includes any decrease inthe rate of growth of a fungal colony. Fungistatic activity may bemanifested by a fungus maintaining its present size or failing tocolonize the surrounding areas. Fungistatic activity may be a result ofinhibition of the fungal reproductive processes. Fungicidal activitygenerally includes, for example, irraditication of a fungus or fungalcolony, killing a fungus or fungal colony or, in one embodiment, adecrease in the mass or size of a fungus or fungal colony.

The term “tetracycline compounds” includes tetracycline family memberssuch as methacycline, sancycline, apicycline, clomocycline,guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline,etamocycline, penimocycline, etc. as well as other tetracyclinecompounds having the characteristic naphthacene A-B-C-D ring structure.Additional tetracycline compounds can be found, for example, in U.S.patent application Ser. No.: 09/234,847, and U.S. Pat. Nos. 5,834,450;5,532,227; 5,789,395; 5,639,742 and German patents DE 28 14 974 and DE28 20 983. The entire contents of the aforementioned applications andpatents are hereby expressly incorporated herein by reference.

Recent research efforts have focused on developing new tetracyclinecompositions effective under varying therapeutic conditions and routesof administration; and for developing new tetracycline analogues whichmight prove to be equal or more effective as antibiotics than theoriginally introduced tetracycline families (See, U.S. Pat. Nos.3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280;4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531).

The term “substituted tetracycline compounds” includes tetracyclinecompounds which have at least one substitution, e.g., at the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 11a, 12 and 12a position, which allows the compoundto perform its intended function, e.g., inhibit the growth of fungus. Inan embodiment, the term “substituted tetracycline compounds” does notinclude unsubstituted tetracycline, minocycline, or doxycycline. In anembodiment, the substituted tetracycline compounds of the invention havelower MIC for Candida fungus (as measured in the assay given in Example2) than doxycycline or minocycline. In one embodiment, the substitutedtetracycline compounds of the invention have MIC's for a fungus which isabout 95% or less, about 90% or less, about 85% or less, about 80% orless, about 75% or less, about 70% or less, about 65% or less, about 60%or less, about 55% or less, about 50% or less, about 45% or less, about40% or less, about 35% or less, about 30% or less, about 25% or less,about 20% or less, about 15% or less, about 10% or less, about 5% orless, or about 1% or less than the MIC of unsubstituted tetracycline,unsubstituted doxycycline, or unsubstituted minocycline for thatparticular fungus. Values and ranges included and/or intermediate withinthe ranges set forth herein are also intended to be within the scope ofthe present invention. For example, a MIC of less than 10% includesMIC's of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc., which are intended tobe included within the range of less than 10%.

Furthermore, the substituted tetracycline compounds also mayadvantageously be less cytotoxic than unsubstituted tetracycline,minocycline, or doxycycline. In one embodiment, the cytotoxicity of thesubstituted tetracycline compounds is about 95% or less, about 90% orless, about 85% or less, about 80% or less, about 75% or less, about 70%or less, about 65% or less, about 60% or less, about 55% or less, about50% or less, about 45% or less, about 40% or less, about 35% or less,about 30% or less, about 25% or less, about 20% or less, about 15% orless, about 10% or less, about 5% or less, or about 1% or less than thecytotoxicity of unsubstituted tetracycline, unsubstituted doxycycline,or unsubstituted minocycline. Values and ranges included and/orintermediate within the ranges set forth herein are also intended to bewithin the scope of the present invention. For example, a cytoxicity ofless than 10% includes cytotoxicities of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, etc. are intended to be included within the range of less than 10%.

The term “substituted tetracycline compound” includes, for example,substituted sancycline compounds, substituted minocycline compounds andsubstituted doxycycline compounds.

Substituted tetracycline compounds used in the methods and compositionsof the invention include compounds of Formula I:

-   -   X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;    -   R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen,        alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen,        or hydrogen;    -   R³, R¹¹ and R¹² are each hydrogen, or a pro-drug moiety;    -   R¹⁰ is hydrogen, a prodrug moiety, or linked to R⁹ to form a        ring;    -   R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl,        aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,        alkyl carbonyloxy, or aryl carbonyloxy;    -   R⁶ and R^(6′) are each independently hydrogen, methylene,        absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or        an arylalkyl;    -   R⁷ is hydrogen, halogen, nitro, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,        amino, arylalkenyl, arylalkynyl, or        —(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a);    -   R⁹ is hydrogen, halogen, nitro, alkyl, alkenyl, alkynyl, aryl,        alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,        amino, arylalkenyl, arylalkynyl, thionitroso(e.g., —N═S), or        —CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);    -   Z is CR^(9d), R^(9e), S, NR^(9b) or O;    -   Z′ is O, S, or NR^(9f);    -   W is CR^(7d)R^(7e), S, NR^(7b) or O;    -   W′ is O, NR^(7f) S;    -   R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(9a), R^(9b), R^(9c),        R^(9d), and R^(9e) are each independently hydrogen, acyl, alkyl,        alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,        alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,        heteroaromatic or a prodrug moiety;    -   R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl,        alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,        alkylamino, or an arylalkyl;    -   R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl; and    -   Y′ and Y are each independently hydrogen, halogen, hydroxyl,        cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,        alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an        arylalkyl, and pharmaceutically acceptable salts thereof.

In an embodiment, the substituted tetracycline compounds used in themethods and compositions of the invention are substituted sancyclinecompounds, e.g., with substitution at the, for example, 2, 5, 6, 7,8, 9,10, 11, 11a, 12, 12a position and/or, in the case of minocycline, 13. Insubstituted sancycline compounds of the invention, R^(2′), R³, R¹⁰, R¹¹,and R¹² are each hydrogen or a prodrug moiety; R^(4′) and R^(4″) areeach alkyl (e.g., lower alkyl, e.g., methyl); X is CR⁶R^(6′); and R²,R⁵, R⁶, R^(6′), and R⁸ are each, generally, hydrogen. In otherembodiments, In an embodiment, the substituted tetracycline compound isa substituted tetracycline (e.g., generally, wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are methyl, R⁵ is hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy); substituteddoxycycline (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) aremethyl, R⁵ is hydroxyl and X is CR⁶R^(6′), wherein R⁶ is methyl andR^(6′) is hydrogen); substituted minocycline (e.g., wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are methyl; R⁵ is hydrogen and X isCR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogen atoms and R⁷ isdimethylamino) or substituted sancycline (wherein R⁴ is NR^(4′)R^(4″),R^(4′) and R^(4″) are methyl; R⁵ is hydrogen and X is CR⁶R^(6′) whereinR⁶ and R^(6′) are hydrogen atoms).

In one embodiment, R⁵is substituted, e.g., not hydrogen or hydroxy. In afurther embodiment R⁵ is an ester (alkcarbonyloxy). In an embodiment, R⁵is an alkyl ester. Examples of R⁵ include alkyl esters such as C₁-C₁₂alkyl, alkenyl, alkynyl, or aryl esters. The alkyl groups may bestraight chains, branched chains, and/or contain rings. Examples ofesters include, but are not limited to, tetracycline esters of ethanoicacid, propanoic acid, pentanoic acid, hexanoic acid, 2-cyclopentaneethanoic acid, cyclopentanoic acid, cycloheptanoic acid, 2-methylpropanoic acid, cyclohexanoic acid, and adamantane 2-carboxylic acid. Inother embodiments, R⁵ is hydrogen.

For 7-substituted tetracycline compounds, R⁹ may be hydrogen. In oneembodiment, R⁷ is substituted or unsubstituted phenyl. Examples of R⁷substituents include all substituents which allow the tetracyclinecompound to perform its intended function, such as but are not limitedto, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

In an embodiment, the phenyl is substituted with at least one alkylgroup, which itself may be, branched, straight chain or alkyl,unsubstituted or substituted (e.g., halogenated). Examples of alkylgroups include, but are not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, etc.

In another embodiment R⁷ is a halogen, e.g., chlorine, bromine, oriodine.

In another embodiment, R⁷ is substituted or unsubstituted heteroaryl.Examples of heteroaromatic groups include both monocyclic and polycyclic(e.g., multicylic rings), such as, but not limited to, furanyl,imidazolyl, benzothiophenyl, benzofuranyl, quinolinyl, isoquinolinyl,pyridinyl, pyrazolyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl,benzoimidazolyl, methylenedioxyphenyl, indolyl, thienyl, pyrimidyl,pyrazinyl, purinyl, pyrazolyl, oxazolyl, isooxazolyl, naphthridinyl,thiazolyl, isothiazolyl, and deazapurinyl. In an embodiment, R⁷ isbenzofuranyl. Examples of substituents include all substituents whichallow the tetracycline compound to perform its intended function, suchas but are not limited to, alkyl, alkenyl, alkynyl, halogen, hydroxyl,alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

In another embodiment, R⁷ is substituted or unsubstituted, branched,straight chain or cyclic alkyl. Examples of substituents include thosewhich allow the substituted tetracycline compound to perform itsintended function, e.g., inhibit the growth of a fungus. Examples ofsubstitutents include, but are not limited to, alkenyl, alkynyl,halogen, hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl,arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, aminoalkyl, trialkylsilyl, arylalkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato, cyano,amino, acylamino, amido, imino, sulfhydryl, alkylthio, sulfate,arylthio, thiocarboxylate, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl, aryl andheteroaryl.

Examples of alkyl R⁷ groups include C₁-C₁₅ groups and C₁-C₁₀ groups.Examples include 2-ethyl pentyl, methyl, ethyl, propyl, pentyl, hexyl,heptyl, etc.

In one embodiment, R⁷ is substituted or unsubstituted alkenyl. Examplesof substituents include all substituents which allow the tetracyclinecompound to perform its intended function, such as but are not limitedto, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

In one embodiment, an alkenyl R⁷ moiety is substituted with asubstituted or unsubstituted cyclic moiety. Cyclic moieties include bothcarbocyclic, heterocyclic, aryl, heteroaryl, cycloalkenyl, andcycloalkyl groups. Examples of cyclic moieties include, for example,cyclobutane, cylopentane, cyclohexane, phenyl, etc. The cyclic moietycan be substituted, e.g., with any substituent listed above for alkenylR⁷ moieties.

In another embodiment, R⁷ is substituted or unsubstituted alkynyl.Examples of substituents include all substituents which allow thetetracycline compound to perform its intended function, such as but arenot limited to, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

The R⁷ alkynyl moiety may be substituted with a substituted orunsubstituted cyclic moiety. Cyclic moieties include both carbocyclic,heterocyclic, aryl, heteroaryl, cycloalkenyl, and cycloalkyl groups.Examples of cyclic moieties include, for example, cyclobutane,cylopentane, cyclohexane, phenyl, etc. The cyclic moiety can besubstituted, e.g., with any substituent listed above for alkynyl R⁷moieties.

Examples of cyclic substituents for alkynyl R⁷ moieties include, but arenot limited to, phenyl, cyclohexyl, p-nitro phenyl, p-methyl phenyl, and1-hydroxy cyclohexane.

In another embodiment. R⁷ is substituted or unsubstituted alkoxy.Examples of substituents include all substituents which allow thetetracycline compound to perform its intended function, such as but arenot limited to, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl. In one embodiment, the alkoxy group is C₁-C₁₀. Inanother embodiment, it is methoxy, ethoxy, propoxy, butoxy, pentoxy,hexoxy, etc.

In another embodiment, the invention pertains to substituted doxycyclinecompounds wherein R⁵ is hydroxy or alkylcarbonyloxy; X is CHR⁶; R⁶ isalkyl (e.g., lower alkyl, e.g., methyl); and R⁸ is hydrogen. R⁷ may behydrogen or alkyl. R² may be hydrogen or alkyl.

In one embodiment, R⁹ is substituted or unsubstituted aryl, e.g.,phenyl, biaryl, heteroaryl (e.g., pyridine, etc.), etc. Examples ofsubstituents include all substituents which allow the tetracyclinecompound to perform its intended function, such as but are not limitedto, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

Other examples of R⁹ include substituted or unsubstituted alkyl (e.g.,methyl, ethyl, propyl, t-butyl, n-butyl, i-butyl, pentyl, etc.)or N₃.

In another embodiment, the invention pertains to methods andcompositions which the substituted tetracycline compound is asubstituted minocycline compound. Examples of these compounds includecompounds wherein X is CR⁶R^(6′); R², R⁵, R⁶, R^(6′), and R⁸ are eachhydrogen, and R⁷ is dimethyl amino.

In an embodiment, R⁹ is substituted or unsubstituted aryl (e.g., phenyl,biaryl (naphthyl, benzofuranyl), heteroaryl, etc.) or araalkyl. Examplesof substituents include, but are not limited to, alkyl, alkenyl,alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl,carboxy, arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl,arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, aminoalkyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, silyl, aminocarbonyl, alkylthiocarbonyl, phosphate,aralkyl, phosphonato, phosphinato, cyano, amino, acylamino, amido,imino, sulfhydryl, alkylthio, sulfate, arylthio, thiocarboxylate,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, azido,heterocyclyl, alkylaryl, aryl and heteroaryl. In one embodiment, thearyl R⁹ moiety is substituted or unsubstituted phenyl.

Other examples of R⁹ moieties include substituted and unsubstituted,cyclic, branched or straight chain alkyl (e.g., C₁-C₁₅, C₁-C₁₀, e.g.,methyl, ethyl, propyl, butyl, pentyl, hexyl, 2-cyclopentane ethyl,etc.). Examples of substituents include those listed above for aryl R⁹moieties.

In another embodiment, R⁹ is substituted or unsubstituted, branched,straight chain or cyclic alkyl. Examples of substituents include thosewhich allow the substituted tetracycline compound to perform itsintended function, e.g., inhibit the growth of a fungus. Examples ofsubstitutents include, but are not limited to, alkenyl, alkynyl,halogen, hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl,arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, aminoalkyl, trialkylsilyl, arylalkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato, cyano,amino, acylamino, amido, imino, sulfhydryl, alkylthio, sulfate,arylthio, thiocarboxylate, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl, aryl andheteroaryl.

Examples of alkyl R⁹ groups include C₁-C₁₅ groups and C₁-C₁₀ groups.Examples include 2-ethyl pentyl, methyl, ethyl, propyl, pentyl, hexyl,heptyl, etc.

In one embodiment, R⁹ is substituted or unsubstituted alkenyl. Examplesof substituents include all substituents which allow the tetracyclinecompound to perform its intended function, such as but are not limitedto, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

In one embodiment, an alkenyl R⁹ moiety is substituted with asubstituted or unsubstituted cyclic moiety. Cyclic moieties include bothcarbocyclic, heterocyclic, aryl, heteroaryl, cycloalkenyl, andcycloalkyl groups. Examples of cyclic moieties include, for example,cyclobutane, cylopentane, cyclohexane, phenyl, etc. The cyclic moietycan be substituted, e.g., with any substituent listed above for alkenylR⁹ moieties.

In another embodiment, R⁹ is substituted or unsubstituted alkynyl.Examples of substituents include all substituents which allow thetetracycline compound to perform its intended function, such as but arenot limited to, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aminoalkyl,arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl, alkylaryl,aryl and heteroaryl.

The R⁹ alkynyl moiety may be substituted with a substituted orunsubstituted cyclic moiety. Cyclic moieties include both carbocyclic,heterocyclic, aryl, heteroaryl, cycloalkenyl, and cycloalkyl groups.Examples of cyclic moieties include, for example, cyclobutane,cylopentane, cyclohexane, phenyl, etc. The cyclic moiety can besubstituted, e.g., with any substituent listed above for alkynyl R⁹moieties.

Furthermore, the substituted tetracycline compounds of the invention canbe substituted with combinations of any one of the substituentsdescribed above or shown in Table 2.

Examples of cyclic substituents for alkynyl R⁹ moieties include, but arenot limited to, phenyl, cyclohexyl, p-nitro phenyl, p-methyl phenyl, and1-hydroxy cyclohexane.

In a further embodiment, R⁹ is substituted or unsubstituted aminoalkyl.Examples of substituents include, but are not limited to, alkyl,alkenyl, alkynyl, and aryl substituents.

In one embodiment, the substituted tetracycline compounds for use in themethods and compositions of the invention have a greater antifungalactivity than unsubstituted doxycycline or minocycline. Both of thesecompounds have Minimum Inhibitory Concentrations (MIC) for C. Albicans,C. giabrata, C. tropicalis, and C. parapsilosis in excess of 64 (Seeexample 2, for the procedure for measuring the MIC).

In another embodiment, the substituted tetracycline compounds of theinvention may have anti-inflammatory activity, e.g., as measured in artrecognized assays.

In another embodiment, the substituted tetracycline compounds of theinvention may be non-antibacterial, e.g., have little or noantibacterial activity. The antibacterial activity of the compounds ofthe invention can be measured using the assay given in Example 5. In anembodiment, a compound is considered to be non-antibacterial if it has aMIC (against bacteria) of 4 μM or greater.

In another embodiment, the invention also pertains to4-dedimethylaminotetracycline compounds with the substituents describedherein or shown in Table 2 (e.g., compounds with the same substituentsas described herein or in Table 2, except at the R⁴ position where theshown dimethylamino group is a hydrogen.)

Examples of substituted tetracycline compounds for use in the methodsand compositions of the invention include the compounds shown in Table2, as well as those shown below:

The substituted tetracycline compounds of the invention can besynthesized using the methods described in Example 1 and in thefollowing schemes. Any novel tetracycline compounds described herein areincluded in the invention as compounds, in addition to methods of usingthem and pharmaceutical compositions containing them.

9-substituted tetracyclines such as 9-cyclopentenyl doxycycline can besynthesized by the method shown in Scheme 1. As in Scheme 1,9- and7-substituted tetracycline compounds can be synthesized by treating atetracycline compound (e.g., doxycycline, 1 A), with sulfuric acid andsodium nitrate. The resulting product is a mixture of the 7-nitro and9-nitro isomers (1 B and 1 C, respectively). The 7-nitro (1 B) and9-nitro (1 C) derivatives are treated by hydrogenation using hydrogengas and a platinum catalyst to yield amines 1 D and 1 E. The isomers areseparated at this time by conventional methods. To synthesize 7- or9-substituted alkenyl derivatives, the 7- or 9-amino tetracyclinecompound (1 E and 1 F, respectively) is treated with HONO, to yield thediazonium salt (1 G and 1 H). The salt (1 G and 1 H) is treated with anappropriate halogenated reagent (e.g., R⁹Br, wherein R⁹ is an aryl,alkenyl, or alkynyl moiety) to yield the desired compound(e.g., inScheme 1, 7-cyclopent-1-enyl doxycycline (1 H) and 9-cyclopent-1-enyldoxycycline (1 I)).

As shown in Scheme 2, tetracycline compounds of the invention wherein R⁷is a carbamate or a urea derivative can be synthesized using thefollowing protocol. Sancycline (2 A) is treated with NaNO₂ under acidicconditions forming 7-nitro sancycline (2 B) in a mixture of positionalisomers. 7-nitrosancycline (2 B) is then treated with H₂ gas and aplatinum catalyst to form the 7-amino sancycline derivative (2 C). Toform the urea derivative (2 E), isocyanate (2 D) is reacted with the7-amino sancycline derivative (2 C). To form the carbamate (2 G), theappropriate acid chloride ester (2 F) is reacted with 2 C.

As shown in Scheme 3, tetracycline compounds of the invention, whereinR⁷ is a heterocyclic (i.e. thiazole) substituted amino group can besynthesized using the above protocol. 7-amino sancycline (3 A) isreacted with Fmoc-isothiocyanate (3 B) to produce the protected thiourea(3 C). The protected thiourea (3 C) is then deprotected yielding theactive sancycline thiourea (3 D) compound. The sancycline thiourea (3 D)is reacted with an α-haloketone (3 E) to produce a thiazole substituted7-amino sancycline (3 F).

7-alkenyl tetracycline compounds, such as 7-alkynyl sancycline (4 A) and7-alkenyl sancycline (4 B), can be hydrogenated to form alkyl7-substituted tetracycline compounds (e.g., 7-alkyl sancycline, 4 C).Scheme 4 depicts the selective hydrogenation of the 7-position double ortriple bond, in saturated methanol and hydrochloric acid solution with apalladium/carbon catalyst under pressure, to yield the product.

In Scheme 5, a general synthetic scheme for synthesizing 7-position arylderivatives is shown. A Suzuki coupling of an aryl boronic acid with aniodosancycline compound is shown. An iodo sancycline compound (5 B) canbe synthesized from sancycline by treating sancycline (5 A) with atleast one equivalent N-iodosuccinimide (NIS) under acidic conditions.The reaction is quenched, and the resulting 7-iodo sancycline (5 B) canthen be purified using standard techniques known in the art. To form thearyl derivative, 7-iodo sancycline (5 B) is treated with an aqueous base(e.g., Na₂CO₃) and an appropriate boronic acid (5 C) and under an inertatmosphere. The reaction is catalyzed with a palladium catalyst (e.g.,Pd(OAc)₂). The product (5 D) can be purified by methods known in the art(such as HPLC). Other 7-aryl and alkynyl tetracycline compounds can besynthesized using similar protocols.

The 7-substituted tetracycline compounds of the invention can also besynthesized using Stille cross couplings. Stille cross couplings can beperformed using an appropriate tin reagent (e.g., R—SnBu₃) and ahalogenated tetracycline compound, (e.g., 7-iodosancycline). The tinreagent and the iodosancycline compound can be treated with a palladiumcatalyst (e.g., Pd(PPh₃)₂Cl₂ or Pd(AsPh₃)₂Cl₂) and, optionally, with anadditional copper salt, e.g., CuI. The resulting compound can then bepurified using techniques known in the art.

The compounds of the invention can also be synthesized using Heck-typecross coupling reactions. As shown in Scheme 6, Heck-typecross-couplings can be performed by suspending a halogenatedtetracycline compound (e.g., 6-iodosancycline, 6 A) and an appropriatepalladium or other transition metal catalyst (e.g., Pd(OAc)₂ and CuI) inan appropriate solvent (e.g., degassed acetonitrile). The substrate, areactive alkene (6 B) or alkyne (6 D), and triethylamine are then addedand the mixture is heated for several hours, before being cooled to roomtemperature. The resulting 7-substituted alkenyl (6 C) or 7-substitutedalkynyl (6 E) tetracycline compound can then be purified usingtechniques known in the art.

To prepare 7-(2′-Chloro-alkenyl)-tetracycline compounds, the followingprocedure can be used. 7-(alkynyl)-sancycline (7 A) is dissolved insaturated methanol and hydrochloric acid and stirred. The solvent isthen removed to yield the product (7 B).

As depicted in Scheme 8,5-esters of 9- substituted tetracyclinecompounds can be formed by dissolving the 9- substituted compounds (8 A)in strong acid (e.g. HF, methanesulphonic acid, andtrifluoromethanesulfonic acid) and adding the appropriate carboxylicacid to yield the corresponding esters (8 B).

As shown in Scheme 9 below, 7 and 9 aminomethyl tetracyclines may besynthesized using reagents such as hydroxymethyl-carbamic acid benzylester.

The term “alkenyl” includes unsaturated aliphatic groups, includingstraight-chain alkenyl groups, branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups, alkenyl substituted cycloalkyl orcycloalkenyl groups, and cycloalkenyl substituted alkyl or alkenylgroups. The term alkenyl further includes alkenyl groups, which canfurther include oxygen, nitrogen, sulfur or phosphorous atoms replacingone or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen,sulfur or phosphorous atoms. In preferred embodiments, a straight chainor branched chain alkenyl group has 10 or fewer carbon atoms in itsbackbone (e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ for branched chain),and more preferably 6 or fewer. Likewise, preferred cycloalkenyl groupshave from 4-7 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure, e.g., cyclopentene orcyclohexene.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone, e.g., oxygen,nitrogen, sulfur or phosphorous atoms. In preferred embodiments, astraight chain or branched chain alkyl has 10 or fewer carbon atoms inits backbone (e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ for branchedchain), and more preferably 6 or fewer. Likewise, preferred cycloalkylshave from 4-7 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)).

The term “aryl” includes aryl groups, including 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like. Aryl groups alsoinclude polycyclic fused aromatic groups such as naphthyl, quinolyl,indolyl, and the like. Those aryl groups having heteroatoms in the ringstructure may also be referred to as “aryl heterocycles”, “heteroaryls”or “heteroaromatics”. The aromatic ring can be substituted at one ormore ring positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromaticmoiety. Aryl groups can also be fused or bridged with alicyclic orheterocyclic rings which are not aromatic so as to form a polycycle(e.g., tetralin).

The terms “alkenyl” and “alkynyl” include unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. Examples of substituents of alkynyl groups include, forexample alkyl, alkenyl (e.g., cycloalkenyl, e.g., cyclohenxenyl), andaryl groups.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto three carbon atoms in its backbone structure. Likewise, “loweralkenyl” and “lower alkynyl” have similar chain lengths.

The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (includingalkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “alkylsulfinyl” include groups which have one or more sulfinyl(SO) linkages, typically 1 to about 5 or 6 sulfinyl linkages.Advantageous alkylsulfinyl groups include groups having 1 to about 12carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkylsulfonyl” includes groups which have one or more sulfonyl(SO₂) linkages, typically 1 to about 5 or 6 sulfonyl linkages.Advantageous alkylsulfonyl groups include groups having 1 to about 12carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkanoyl” includes groups having 1 to about 4 or 5 carbonylgroups. The term “aroyl” includes aryl groups, such as phenyl and othercarbocyclic aryls, which have carbonyl substituents. The term “alkaroyl”includes aryl groups with alkylcarbonyl substituents, e.g.,phenylacetyl.

The structures of some of the tetracycline compounds of this inventioninclude asymmetric carbon atoms. The isomers arising from the chiralatoms (e.g., all enantiomers and diastereomers) are included within thescope of this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis.

The invention also pertains to methods of treating fungal associateddisorders in a subject, by administering to the subject an effectiveamount of a substituted tetracycline compound such that the subject istreated. The substituted tetracycline compound may, in one embodiment,be a compound of formula (I) or any one of the compounds depicted inTable 2.

The language “effective amount” of the tetracycline compound is thatamount necessary or sufficient to inhibit the growth of fungus or treata fungus associated disorder, e.g., in an animal or in a plant, e.g.,and prevent the various morphological and somatic symptoms of a fungalassociated disorder. The effective amount can vary depending on suchfactors as the size and weight of the subject, the type of disorder, orthe particular substituted tetracycline compound. For example, thechoice of the substituted tetracycline compound can affect whatconstitutes an “effective amount”. One of ordinary skill in the artwould be able to study the aforementioned factors and make thedetermination regarding the effective amount of the substitutedtetracycline compound without undue experimentation. An in vivo assay asdescribed in Example 4 below or an assay similar thereto (e.g.,differing in choice of cell line or type of illness) also can be used todetermine an “effective amount” of a tetracycline compound. Theordinarily skilled artisan would select an appropriate amount of atetracycline compound for use in the aforementioned in vivo assay. In anembodiment, the effective amount of the tetracycline is effective totreat a mammal suffering from a fungal associated disorder which isassociated with a fungus from the genus Candida.

The term “subject” any organism which may benefit from the inhibition ofa fungus or which is capable of having a fungal associated disorder.Examples of subjects include not only animals, such as mammals, birds,fish, etc., but plants which may be adversely effected by the presenceof a fungus.

The term “mammal” includes, but is not limited to, ruminants (e.g.,cattle and goats), mice, rats, hamsters, dogs, cats, horses, pigs,sheep, lions, tigers, bears, monkeys, chimpanzees, and, in a preferredembodiment, humans. The mammal may be immunocompetent orimmunocompromised, e.g., suffering from an immunodeficiency. Forexample, the mammal may have AIDS or may have previously or concurrentlyundergone chemotherapy. In another embodiment, the mammal may be elderlyor young. The mammal may or may not be suffering from a fungalassociated disorder. The tetracycline compounds may be administered to amammal susceptible to a fungal associated disorder to prevent theoccurrence of the disorder.

The language “fungal associated disorder” includes disorders which arerelated to the presence of fungus in a subject. Examples of fungalassociated disorders include both topical fungal infections caused by,e.g., Candida, and dermatophytes such as Trichophyton, Microsporum orEpidermophyton, or in mucosal infections caused by Candida albicans(e.g., oral thrush and vaginal candidiasis). The substitutedtetracycline compounds of the invention are also useful for treatment ofsystemic fungal infections caused by, for example, Candida albicans,Cryptococcus neoformans, Aspergillus flavus, Aspergillus funigatus,Coccidioides, Paracoccidioides, Histoplasma or Blastomyces. Thesubstituted tetracycline compounds of the invention may be useful fortreating fungal infections in immunocompromised patients such aspatients with viral infections such as AIDS, CMV, and influenza, cancerpatients receiving chemotherapy or radiotherapy, transplant patientsreceiving antirejection agents, and patients that have received toxicchemicals, metals and radiation exposure.

Examples of fungal associated disorders in animals include systemicinfections, such as histoplasmosis, systemic candidiasis, aspergillosis,blastomycosis, coccidioidomycosis, paracoccidioidomycosis, andcryptococcosis, and superficial fungal disorders, such as dermatophyte(ringworm) infections, for example, tinea pedis (athlete's foot) andtinea cruris (jock itch), candidiasis, and actinomycosis. Anotherexample of a fungal associated disorder include mycoses, which may becaused by fungi which are opportunists, rather than pathogens. Examplesof fungi which may cause mycoses include candidiasis, aspergillosis,phycomycosis, nocardiosis, and cryptococcosis.

Other fungal associated disorders include aspergillosis, candidosis,chromomycosis, coccidioidiocycosis, cryptocococcosis,entomophthoromycosis, epizootic lymphangitis, geotrichosis,histoplasmosis, mucormycosis, mycetoma, north american blastomycosis,oomycosis, paecilimycosis, penicilliosis, rhinosporidiosis, andsprotrichiosis in animals. In an embodiment, the substitutedtetracycline compounds of the invention can be included in feed for thelivestock, such that normal consumption of said feed provides about 1 mgto about 200 mg of at least one of the substituted tetracyclinecompounds of the invention per kg of animal per day.

The invention also pertains to a pharmaceutical composition containingan effective amount of a tetracycline compound to treat or prevent afungal associated disorder in a subject, e.g., a mammal, and apharmaceutically acceptable carrier. The tetracycline compound may be asubstituted tetracycline, a compound of formula (I), or a compounddepicted in Table 2.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the substituted tetracyclinecompound(s), and which allows the substituted tetracycline compounds toperform their intended function, e.g., treating a fungal associatedstate or preventing a fungal associated disorder. Examples of suchcarriers include solutions, solvents, dispersion media, delay agents,emulsions and the like. The use of such media for pharmaceuticallyactive substances are well known in the art. Any other conventionalcarrier suitable for use with the tetracycline compounds of the presentinvention are included.

For example, one or more substituted tetracycline compounds of theinvention may be administered alone to a subject, or more typically acompound of the invention will be administered as part of apharmaceutical composition in mixture with conventional excipient, i.e.,pharmaceutically acceptable organic or inorganic carrier substancessuitable for parenteral, oral or other desired administration and whichdo not deleteriously react with the active compounds and are notdeleterious to the recipient thereof. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

At least many of the substituted tetracycline compounds of the inventionsuitably may be administered to a subject in a protonated andwater-soluble form, e.g., as a pharmaceutically acceptable salt of anorganic or inorganic acid, e.g., hydrochloride, sulfate, hemi-sulfate,phosphate, nitrate, acetate, oxalate, citrate, maleate, mesylate, etc.Also, where an appropriate acidic group is present on a compound of theinvention, a pharmaceutically acceptable salt of an organic or inorganicbase can be employed such as an ammonium salt, or salt of an organicamine, or a salt of an alkali metal or alkaline earth metal such as apotassium, calcium or sodium salt.

Therapeutic compounds can be administered to a subject in accordancewith the invention by any of a variety of routes. Topical (includingtransdermal, buccal or sublingual), oral, and parenteral (includingintraperitoneal, subcutaneous, intravenous, intradermal or intramuscularinjection) are generally preferred.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof. The tetracycline compounds may also be formulatedsuch that the compound is released over an extended period of time.

For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions as well as suspensions, emulsions,or implants, including suppositories. Substituted tetracycline compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

For topical applications, the substituted tetracycline compound(s) canbe suitably admixed in a pharmacologically inert topical carrier such asa gel, an ointment, a lotion or a cream. Such topical carriers includewater, glycerol, alcohol, propylene glycol, fatty alcohols,triglycerides, fatty acid esters, or mineral oils. Other possibletopical carriers are liquid petrolatum, isopropylpalmitate, polyethyleneglycol, ethanol 95%, polyoxyethylene monolauriate 5% in water, sodiumlauryl sulfate 5% in water, and the like. In addition, materials such asanti-oxidants, humectants, viscosity stabilizers and the like also maybe added if desired.

The actual preferred amounts of active substituted tetracyclinecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

For example, a suitable effective dose of one or more compounds of theinvention will be in the range of from 0.01 to 100 milligrams perkilogram of body weight of recipient per day, preferably in the range offrom 0.1 to 50 milligrams per kilogram body weight of recipient per day,more preferably in the range of 1 to 20 milligrams per kilogram bodyweight of recipient per day. The desired dose is suitably administeredonce daily, or several sub-doses, e.g. 2 to 5 sub-doses, areadministered at appropriate intervals through the day, or otherappropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

In a still further aspect, the substituted tetracycline compounds of thepresent invention (e.g., substituted tetracycline compounds, e.g.,compounds of formula (I), or compounds shown in Table 2) can also beadvantageously used in agricultural compositions, for example,compositions for plants and seeds to treat or prevent a variety of plantpathogenic fungi, including rusts, mildews, and molds. Generally, thecompounds of the present invention are dispensed in the form of dustingpowders, granules, seed dressings, aqueous solutions, dispersions oremulsions, dips, sprays, aerosols or smokes. Compositions may also besupplied in the form of dispersible powders, granules or grains, orconcentrates for dilution prior to use. Such compositions may containsuch conventional carriers, diluents or adjuvants as are known andacceptable in agriculture and horticulture, and they are manufactured inaccordance with conventional procedures. The compositions typicallycontain from 0.01 to 10 wt %, preferably 0.1 to 1 wt. % of the activeingredient. The compositions may also incorporate other activeingredients, for example, compounds having herbicidal or insecticidalactivity or a further fungicide. The compounds and compositions can beapplied in a number of ways, for example, they can be applied directlyto the plant foliage, stems, branches, seeds or roots or to the soil orother growing medium and they may be used not only to eradicate disease,but also prophylactically to protect the plants or seeds from attack.For field use, likely application rates of active ingredient are about100 to 10,000 g/acre. The substituted tetracycline compounds of theinvention can also be formulated for use in cleaning supplies, e.g., toprevent a growth of a fungus, or to kill or stop the growth of a fungus

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not to be construed as limitations thereof.

EXEMPLIFICATION OF THE INVENTION Example 1

Synthesis of Tetracycline Compounds

The following example discusses methods of synthesizing the substitutedtetracycline compounds of the invention. One of ordinary skill in theart will be able to use the presented examples and/or art recognizedtechniques to synthesize the compounds of the invention.

Experimental

Melting points were taken on a Mel-Temp capillary melting pointapparatus and are uncorrected. Nuclear magnetic resonance (¹H NMR)spectra were recorded at 300 MHz on a Bruker Avance spectrometer. Thechemical shift values are expressed in δ values (ppm) relative totetramethylsilane or 3-(trimethylsilyl)-1-propanesulfonic acid, sodiumsalt, as either an internal or external standard using CDCl₃, DMSO-d₆,or MeOH-d₄ as the solvent. Column chromatography was performed accordingto the method of Still using Baker “flash” grade silica gel (40 μm) thatwas treated with a saturated solution of Na₂EDTA, washed with water,filtered and dried in an oven at 130° C. for three hours prior to use.Analytical TLC separations employed the use of 0.25 mm silica gel plateswith florescence indicator obtained from J.T. Baker Chemical Co.,Phillipsburg, N.J., that were pretreated by immersion into a saturatedsolution of Na₂EDTA for five minutes and reactivated at 130° C. forthree hours. Solvent systems used were as follows: 50:50:5 CHCl₃/MeOH/5%Na₂EDTA (lower phase) (I), 65:20:5, CHCl₃/MeOH/Na₂EDTA (lower phase)(II). Visualization of TLC was accomplished by 0.5% aqueous Fast Blue BBsalt and heating at 130° C. for 5 minutes. Analytical HPLC was performedon a Waters Bondapak C18 reverse phase column by using two Varian SD 100HPLC pumps at a 1.6 mL/min flow rate controlled by software. Detectionwas by UV absorption with Model 441 absorbance detector operating at 280nm. Mobile phases used followed a linear gradient from 30% to 100%methanol over 30 minutes at 1.6 mL/min flow rate followed by isocraticelution with MeOH; solvent system A: 0.02 M Na₂HPO₄+0.001 M Na₂EDTAadjusted to pH 4.5 with H₃PO₃; solvent system B: 100% MeOH.Semipreparative HPLC separations used a Waters semipreparative C18reverse-phase column at a flow rate of 6.4 mL/min. Low and highresolution mass spectra were performed on a PE Mariner spectrometer(Nelson el al., J. Med. Chem. (1993) 36(3):374).

7 lodo Sancycline

One gram of sancycline was dissolved in 25 mL of TFA (trifluoroaceticacid) that was cooled to 0° C. (on ice). 1.2 equivalents ofN-iodosuccinimide (NIS) was added to the reaction mixture and reactedfor forty minutes. The reaction was removed from the ice bath and wasallowed to react at room temperature for an additional five hours. Themixture was then analyzed by HPLC and TLC, was driven to completion bythe stepwise addition of NIS. After completion of the reaction, the TFAwas removed in vacuo and 3 mL of MeOH was added to dissolve the residue.The methanolic solution was the added slowly to a rapidly stirringsolution of diethyl ether to form a greenish brown precipitate. The7-iodo isomer of sancycline was purified by treating the 7-iodo productwith activated charcoal, filtering through Celite, and subsequentremoval of the solvent in vacuo to produce the 7-isomer compound as apure yellow solid in 75% yield.

MS(M+H) (formic acid solvent) 541.3.

\Rt: Hypersil C18 BDS Column, 11.73

¹H NMR (Methanol d4-300 MHz) δ7.87-7.90 ( d, 1H), 6.66-6.69 (d, 1H),4.06 (s, 1H), 2.98 (s, 6H), 2.42 (m, 1H), 2.19 (m, 1H), 1.62 (m, 4H),0.99 (m, 2H)

7-(2′,5′ Dimethyl-Phenyl Sancycline

7-iodosancycline (0.28 mM), Pd(OAc)₂ and 10 mL of MeOH are added to aflask with a stir bar and the system degassed 3× using argon. Na₂CO₃(0.8 mM) dissolved in water and argon degassed is added via syringe isadded along with 2,5-dimethylphenyl boronic acid (0.55 mM) in MeOH thatwas also degassed. The reaction was followed by HPLC for 2 hours andcooled to room temperature. The solution was filtered, and dried toproduce a crude mixture. The solid was dissolved in dimethylformamideand injected onto a preparative HPLC system using C18 reverse-phasesilica. The solvent was removed in vacuo to yield the product plussalts. The salts were removed by extraction into 50:25:25 water,butanol, ethyl acetate and dried in vacuo. This solid was dissolved inMeOH and the HCl salt made by bubbling in HCl gas.

7-(Hexynyl)-Sancycline

7-1-Sancycline (1 gm, 1.86 mmol ), taken in 25 mL of acetonitrile wasdegassed and purged with nitrogen (three times). To this suspensionPd(OAc)₂ (20 mg, 0.089 mmol), CuI (10 mg, 0.053 mmol), (o-tolyl)₃P (56mg, 0.183 mmol) were added and purged with nitrogen for few minutes.1-Hexyne (3.72 mmol) and triethylamine (1 mL) were added to thesuspension. It was turned into a brown solution upon addition of Et₃N.The reaction mixture was then heated to 70° C. for 3 hours. Progress ofthe reaction was monitored by HPLC. It was then cooled down to roomtemperature and was filtered through celite. Evaporation of the solventgave a brown solid, which was then purified on preparative HPLC to givea yellow solid. The structure of this compound has been characterizedusing 1H NMR, HPLC, and MS.

9-(4′-Fluorophenylethyl)-Minocycline

9-(4-Fluorophenylethynyl)-minocycline (1 mmol) was taken in saturatedsolution of MeOH/HCl. To this solution 10% Pd/C was added and wassubjected to hydrogenation at 50 psi for 12 hrs. It was then filteredthrough celite. The solvent was evaporated to give a yellow powder.Finally, it was precipitated from MeOH/diethylether. The structure ofthis compound has been characterized using 1H NMR, HPLC, and MS.

9-(2′, 5′-Dimethylphenyl) Minocycline

In a clean, dry reaction vessel, was placed 9-iodominocycline (0.762mmoles) bis HCl salt, palladium (II) acetate (0.076 mmoles) along with10 ml of reagent grade methanol. The solution was immediately purged,with stirring, with a stream of argon gas for approximately 5 minutes.The reaction vessel was brought to reflux and to it was sequentiallyadded via syringe 2M potassium carbonate solution, followed by asolution of 2,5-dimethylphenyl boronic acid (1.53 mmoles) in 5 ml ofreagent DMF. Both of these solutions were previously degassed with argongas for approximately 5 minutes. The reaction was heated for 45 minutes,the progress was monitored via reverse phase HPLC. The reaction wassuctioned filtered through a pad of diatomaceous earth and washed thepad with DMF. The filtrates were reduced to an oil under vacuum andresidue treated with t-butylmethyl ether. Crude material was purifiedvia reverse phase HPLC on DVB utilizing a gradient of water andmethanol/acetonitrile containing 1.0% trifluoroacetic acid.

Example 2

Antifungal Activity of Substituted Tetracycline Compounds

Antifungal activity of the tetracyclines was determined by a brothmicrodillution technique following NCCLS (1997) Standards. Assays weresetup using a Tecan Genesis robotic workstation. All drugs weredissolved in 10% DMSO. Drug concentration ranged from 0.125 to 64 μg/mLin 2 fold serial dilutions. Each tetracycline was tested at 10concentrations ranging from 0.125 to 64 μg/mL. The compounds were testedfor their antifungal activity against Candida albicans (ATCC#90028). Thefinal concentration of DMSO was kept below 1%. Checkerboard analysis ofthe initial hits will be performed to better determine the activity ofthe compound.

The strains tested include those listed in Table 1. TABLE 1 GenusSpecies ATCC/FGSC # Aspergillus fumigatus ATCC 13073 (Fresenius)Aspergillus nidulans FGSCA991 (wt) Candida albicans ATCC90028 Candidaalbicans PCI-1 Candida albicans PCI-17 Candida albicans ATCC 36082Candida glabrata ATCC 90030 Candida guilliermondii ATCC 14242 Candidakrusei ATCC 96685 Candida krusei ATCC 90878 Candida lusitaniae ATCC24347 Candida parapsilosis ATCC 22109 Candida tropicalis ATCC 14246Candida tropicalis ATCC 28707 Cryptococcus neoformans ATCC 90012Cryptococcus neoformans ATCC 90013 Issatchenkia orientalis ATCC 6258Neurospora crassa FGSC853

The results are shown in Table 2. For each compound, * represents goodantifungal activity against the particular fungus, ** represents verygood inhibition of the fungus, and *** represents excellent inhibitionof a particular fungus. TABLE 2 As- As- Sac- In As- per- per- C. Can-Can- Can- Can- Can- Can- Crypto- charo- Vitro per- gillus gillus dub-dida dida dida Can- dida dida dida coccus I. myces Cyto- gillus fumi-fer- lin- albi- gla- guiller- dida lusi- para- tropic- neo- orient-cere- toxi- ID STRUCTURE flavus getus reus iensis cans brata mondilkrusei tanise psilosis alis formans sils visies city A

* ** B

** ** C

* ** D

* ** E

* * * ** F

** ** G

** ** ** ** *** ** ** H

** ** I

** ** J

* ** K

** ** L

* ** M

** ** N

** ** O

** ** P

* * ** Q

* * ** R

** ** S

** ** T

** ** U

* ** V

** ** W

* ** X

* ** Y

*** ** Z

* ** AA

* ** AB

** ** AC

* ** AD

** * AE

*** ** AF

** ** AG

* ** AH

*** ** AI

* ** AJ

** ** AK

* ** AL

* ** AM

** ** AN

** ** AO

* ** AP

* ** AQ

** ** AR

** ** *** ** *** *** *** ** AS

** *** ** AT

** ** AU

* ** AV

* * AW

** ** ** *** ** ** ** ** ** *** ** AX

** ** ** AY

** ** AZ

** ** BA

* ** ** * * ** BB

** ** ** ** ** * ** BC

** *** ** ** ** BD

** ** BE

** ** ** *** *** ** *** *** ** ** BF

* ** * *** *** ** *** *** ** ** BG

** ** BH

* * * ** ** BI

** ** * ** *** *** ** ** ** ** BJ

** ** BK

** ** BL

** ** *** ** ** BM

** ** BN

** ** ** BO

* ** BP

** * BQ

** ** BR

** ** ** ** ** * ** * *** ** BS

** ** BT

** ** ** ** *** ** *** *** ** BU

** ** ** *** ** ** ** ** BV

* ** BW

*** ** BX

** ** ** *** ** ** ** *** ** ** ** BY

* ** ** * * * ** BZ

* ** CA

* ** CB

** ** CC

** ** ** CD

* * CE

** ** ** ** ** ** ** CF

** * CG

** ** CH

** * ** * ** ** ** CI

* ** CJ

** *** ** ** * ** ** ** ** ** CK

* ** CL

* ** ** ** *** *** *** ** ** CM

** * * * ** * ** ** CN

* ** CO

** ** CP

*** ** CQ

* * * ** * ** CR

*** ** CS

* ** CT

* ** CU

* ** CV

*** ** CW

** ** CX

** ** CY

** ** CZ

* ** DA

* ** DB

* ** DC

* ** DD

** ** DE

* ** DF

** *** ** DG

** *** ** DH

*** *** ** DI

** ** DJ

* * DK

* ** DL

* ** DM

* * DN

** ** ** DO

* ** DP

* * * DQ

** ** DR

*** ** DS

* ** **

Example 3

Mammalian Cytotoxicity Assay

COS-1 and CHO cell suspensions are prepared, seeded into 96-well tissueculture treated black-walled microtiter plates (density determined bycell line), and incubated overnight at 37° C., in 5% CO₂ andapproximately 95% humidity. The following day serial dilutions of drugare prepared under sterile conditions and transferred to cell plates.Cell/Drug plates are incubated under the above conditions for 24 hours.Following the incubation period, media/drug is aspirated and 50 μl ofResazurin is added. Plates are then incubated under the above conditionsfor 2 hours and then in the dark at room temperature for an additional30 minutes. Fluorescence measurements are taken (excitation 535 nm,emission 590 nm). The IC₅₀ (concentration of drug causing 50% growthinhibition) is then calculated. The cytotoxcity of both unsubstitutedminocycline and doxycycline were found to be greater than 25.

Example 4

In vitro Anti-Bacterial Activity Assay

The following assay is used to determine the efficacy of thetetracycline compounds against common bacteria. 2 mg of each compound isdissolved in 100 μl of DMSO. The solution is then added tocation-adjusted Mueller Hinton broth (CAMHB), which results in a finalcompound concentration of 200 μg per ml. The tetracycline compoundsolutions are diluted to 50 μL volumes, with a test compoundconcentration of 0.098 μg/ml. Optical density (OD) determinations aremade from fresh log-phase broth cultures of the test strains. Dilutionsare made to achieve a final cell density of 1×10⁶ CFU/ml. At OD=1, celldensities for different genera should be approximately: E. coli 1 × 10⁹CFU/ml S. aureus 5 × 10⁸ CFU/ml Enterococcus sp. 2.5 × 10⁹ CFU/ml

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the tetracycline compound that inhibits growth.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A pharmaceutical composition comprising an effective amount of asubstituted tetracycline compound to treat a fungal associated disorderin a subject and a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 110, wherein said effective amountis effective to treat histoplasmosis, systemic candidiasis,aspergillosis, blastomycosis, coccidioidomycosis,paracoccidioidomycosis, cryptococcosis, dermatophyte infections, tineapedis, tinea cruris, candidiasis, actinomycosis, mycoses, aspergillosis,candidosis, chromomycosis, entomophthoromycosis, epizootic lymphangitis,geotrichosis, histoplasmosis, mucormycosis, mycetoma, north americanblastomycosis, oomycosis, paecilimycosis, penicilliosis,rhinosporidiosis, or sprotrichiosis.
 3. The pharmaceutical compositionof claim 110, wherein said substituted tetracycline compound is of theformula:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O; R², R^(2′), R^(4′), andR^(4″) are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety; R⁴ is NR^(4′)R^(4″),alkyl, alkenyl, alkynyl, hydroxyl, halogen, or hydrogen; R³, R¹¹ and R¹²are each hydrogen, or a pro-drug moiety; R¹⁰ is hydrogen, a prodrugmoiety, or linked to R⁹ to form a ring; R⁵ is hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy; R⁶ and R^(6′) areeach independently hydrogen, methylene, absent, hydroxyl, halogen,thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; R⁷is hydrogen, halogen,nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, arylalkyl, amino, arylalkenyl, arylalkynyl, or—(CH₂)₀₋₃NR^(7c)C(═W′)WR^(7a); R⁹ is hydrogen, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, thionitroso(e.g., —N═S), or—(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a); Z is CR^(9d)R^(9e), S, NR^(9b)or O; Z′ isO, S, or NR^(9f); W is CR^(7d)R^(7e), S, NR^(7b) or O; W′ is O, NR^(7f)S; R^(7a),R^(7b),R^(7c),R^(7d),R^(7e),R^(9a),R^(9b),R^(9c),R^(9d), andR^(9e) are each independently hydrogen, acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety; R⁸ is hydrogen,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and Y′ and Yare eah independently hydrogen, halogen, hydroxyl, cyano, sulfhydryl,amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl, and pharmaceuticallyacceptable salts thereof.